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The Effect of Human Placenat Derived Mesenchymal-like Stem Cells(HPMSCs) and VEGF Accelerating Wound Healing of Ischemic Random Skin Flaps

Author: LiuZhiHui
Tutor: GaoWenXin;ZhouYanMin
School: Jilin University
Course: Clinical Stomatology
Keywords: Human placental source of mesenchymal stem cells Vascular endothelial growth factor Flap
CLC: R622
Type: PhD thesis
Year: 2009
Downloads: 105
Quote: 0
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Abstract


The purpose of this study is to explore the human placental source of mesenchymal stem cells (HPMSC S ) tell the wound healing process plays a key role in the wound repair cells --- vascular endothelial cell differentiation; build PIRE2-EGFP -VEGF bicistronic eukaryotic plasmid expression vector, and use of liposomal transfection of the eukaryotic plasmid expression vector was transfected into HPMSC S within; explore HPMSC by intraperitoneal injection route S ability to carry the target gene trauma local homing expansion HPMSC S itself promote wound repair and its mechanism of action, and whether it is with VEGF in the wound healing process. synergies, exploring the healing of flaps HPMSC S and VEGF over-proportional. In this study, first by density gradient centrifugation from discarded placental tissue separation and culture of mesenchymal stem cells and flow analyzer its surface markers to analyze the results show that the cells and bone marrow source mesenchymal stem cell's surface signs basically the same; draw a growth curve, and results show that the cell line with the basic characteristics of stem cells; In order the identification HPMSC S differentiation capacity, we were into fat, osteogenic induction identification, the results show that the cells complete with multiple differentiation potential. Identified HPMSC S Jiaruneipi cells induced to differentiate to endothelial cells induced differentiation culture system. The research results show that S HPMSC sustain the culture medium, the morphology of fibroblast-like \Specific surface marker of mature endothelial cells vWF in uninduced HPMSC S is not expressed, but in such endothelial-like cells positive for expression. Flow cytometry showed that the endothelial cell surface markers of CD31, of CD34 expression levels than before induction of increased Dil-Ac-LDL uptake study found that, after the induction of endothelial-like cells have the ability to uptake of Dil-Ac-LDL . Described above, the induction of the experimental program is capable of inducing HPMSC S endothelial cell phenotype appears, its phenotypic characteristics has become even more evident as the induction time prolonged, prompted HPMSC S have the potential to differentiate into endothelial cells in vitro under specific conditions. Order to study HPMSC S Can act as transporters of the VEGF gene, the experimental first constructed bicistronic PIRE2-EGFP-VEGF eukaryotic plasmid expression vector, and liposomal transfection method used Lipofectin liposome success the 165 the eukaryotic expression the carrier PIRES2-EGFP-VEGF , onto HPMSC S in the, green fluorescent microscope observation, we can see the green fluorescent protein EGFP expression Step EGFP has been successfully transferred to the target cells. Prove liposome-mediated PIRES2-EGFP-VEGF 165 transfection HPMSC S is feasible. The transfected cells by G418, a large number of cell clones, rather than the transfected cells 100% death, suggesting that VEGF gene to transfection HPMSC S effect and G418 selection effects are better . Western blot analysis of VEGF (Western-blot technology) found that there is a strong expression of VEGF, and the empty vector transfected group lowly. Biological activity of VEGF by MTT assay: target gene transfection HPMSC S biological activity of VEGF expression compared with empty vector group and the control group was significantly higher than that of the control, the no-load group group, these conclusions prompt: on the one hand, VEGF 165 HPMSC S strong expression was correct modified, HPMSC S can be used as gene therapy The carrier cell; the other hand, prompted HPMSC S may have endocrine VEGF functionality, but the effect is weak. In order to prevent exogenous gene transfection HPMSC S appear gene mutations, loss of multiple differentiation potential, we transfected exogenous genes HPMSC S In view of the multi- The results show that transfection HPMSC S still maintain the characteristics of stem cells to the induction of differentiation given. Finally, the experiments will mark BrdU carry the target gene pIRES2-EGFP-VEGF 165 of HPMSC S injection in rat peritoneal flap anti-BrdU immunohistochemistry observed HPMSC S migratory homing characteristics flap distal ischemic area of ??the experimental group visible the BrdU labeling HPMSC S widely distributed, mostly distributed in the table below the epidermis and dermis layer around the neovascularization, indicating HPMSC S the characteristics have traumatic ischemic area of ??the epidermis and dermis below homing expansion, and may differentiate into endothelial cells and promote angiogenesis. Flap immunohistochemical staining and microvessel counts found to carry the target gene and empty vector HPMSC S group as well as a simple application of the VEGF group flap leather below the epidermis seen a lot of yellow dye particles, said Mingpi flap dermis below the table that a large number of endothelial cells and angiogenesis in the cortex, but the empty vector fewer than carrying purposes genome and VEGF group, carrying the largest number of purposes genome yellowish discoloration of granulosa cells, which indicates that VEGF and HPMSC S < / sub> joint role in promoting angiogenesis than simple HPMSC S or VEGF role, indicating that VEGF and HPMSC S synergy VEGF can promote HPMSC S < / sub> to the endothelial cell differentiation, proliferation, promote angiogenesis HPMSC S is likely secreted through endocrine or next to promote angiogenesis factor indirectly promote angiogenesis. The HE staining histopathological examination also confirmed that carry the target gene pIRES2-EGFP-VEGF flaps below 165 HPMSC S cell components and neovascularization are up, and carry the empty carrier HPMSC S flap below the cell-rich ingredients, but the number of blood vessels is less than the carrying purposes genome and VEGF group, which also confirmed, HPMSC S to repair cell phenotype differentiation features, but its pro-angiogenic effect below VEGF and HPMSC S combined effects and the simple application of VEGF. The survival rate detection: carrying the target gene HPMSC S promote flap into the highest rate, followed by VEGF group than those in the control group again for the no-load group, the three obviously promote flap survival role.

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CLC: > Medicine, health > Surgery > Plastic Surgery (repair surgery ) > Plastic surgery school
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